S-Adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3 3 1 1) in mammalian cells and certain parasites [e.g., Leishmania, Plasmodium, Trypansoma] plays a key role in controlling the intracellular levels of AdoHcy by catalyzing its metabolism to homocysteine (Hcy) and adenosine (Ado). By controlling intracellular levels of AdoHcy, this enzyme plays a pivotal role in regulating S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases which are crucial for the viability of mammalian cells and parasites (e.g., AdoMet-dependent methyltransferases in parasites are involved in mRNA capping and in trans-splicing). When these biochemical processes are inhibited by elevating intracellular levels of AdoHcy using known inhibitors of human AdoHcy hydrolase, antiparasitic effects were observed in vitro and in vivo. These results suggest that, if specific inhibitors of parasite AdoHcy hydrolases could be designed, they would have clinical potential as antiparasitic agents. Crucial for the clinical success of these compounds would be their lack of inhibitory effects on human AdoHcy hydrolase, thus, minimizing toxic effects to mammalian cells. Recently, our laboratory has cloned and overexpressed AdoHcy hydrolases from Leishmania (L) donovani and Trypansoma (T) cruzi and differences in their binding of NAD + compared to the human enzyme (i.e., the Kd for binding of NAD+ to human AdoHcy hydrolase is 120 nM compared to Kd values of approx 1-2 uM for the parasite enzymes) were observed. These differences in NAD+ affinity explain why 3'-deoxyadenosine (3'- deoxy-Ado) is a potent inhibitor of the L donovani and T cruzi enzymes but it has no effect on the human AdoHcy hydrolase. Based on these important new discoveries, we plan during the next grant period to (i) optimize the structural features of 3'-deoxy-Ado for binding to the NAD+ binding site of L donovani, T cruzi, as well as Plasmodium (P) falciparium, AdoHcy hydrolases, (ii) to elucidate the structural basis for the differences in the binding of NAD + to the human and parasite enzymes, and (iii) to elucidate the relationships of structure, catalytic activity and susceptibility to inhibition and their differences between the human and parasite enzymes. The outcome of this research program should be the identification of specific inhibitors of parasite AdoHcy hydiolases that have clinical potential as antiparasitic agents.